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Notum Is Required for Neural and Head Induction via
Wnt Deacylation, Oxidation, and InactivationGraphical Abstract
Highlights
d Notum is an extracellular Wnt deacylase
d Acylation maintains Wnt folding and its active monomeric
conformation
d Notum in neuroectoderm is required for vertebrate neural and
head induction
d Distinct Wnt inactivation mechanisms by Notum and Tiki
orchestrate brain development
Zhang et al., 2015, Developmental Cell 32, 719–730March 23, 2015 ª2015 Elsevier Inc.http://dx.doi.org/10.1016/j.devcel.2015.02.014
Authors
XinjunZhang,Seong-MoonCheong, ...,
Jose Garcia Abreu, Xi He
In Brief
Wnt proteins require a lipid modification
for receptor binding and activity. Zhang
and Cheong et al. report that Notum, a
secreted Wnt antagonist, is a Wnt
deacylase that inactivates Wnt proteins
and is required for vertebrate brain
development. Deacylated Wnt forms
oxidized-oligomers, suggesting that
acylation is essential for Wnt structure.
Accession Numbers
KP781855
KP781856
KP781857
Developmental Cell
Article
Notum Is Required for Neural and Head Inductionvia Wnt Deacylation, Oxidation, and InactivationXinjun Zhang,1,4 Seong-Moon Cheong,1,4 Nathalia G. Amado,1 Alice H. Reis,2 Bryan T. MacDonald,1 Matthias Zebisch,3
E. Yvonne Jones,3 Jose Garcia Abreu,2 and Xi He1,*1Department of Neurology, The F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston,
MA 02115, USA2Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil3Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK4Co-first author
*Correspondence: [email protected]://dx.doi.org/10.1016/j.devcel.2015.02.014
SUMMARY
Secreted Wnt morphogens are essential for em-bryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor bindingand activity. Notum is a secreted Wnt antagonistthat belongs to the a/b hydrolase superfamily, butits mechanism of action and roles in vertebrateembryogenesis are not fully understood. Here, wereport that Notum hydrolyzes the Wnt palmitoleoy-late adduct extracellularly, resulting in inactivatedWnt proteins that form oxidized oligomers incapableof receptor binding. Thus, Notum is aWnt deacylase,and palmitoleoylation is obligatory for the Wnt struc-ture that maintains its active monomeric conforma-tion. Notum is expressed in naive ectoderm and neu-ral plate in Xenopus and is required for neural andhead induction. These findings suggest that Notumis a prerequisite for the ‘‘default’’ neural fate andthat distinct mechanisms of Wnt inactivation by theTiki protease in the Organizer and the Notum de-acylase in presumptive neuroectoderm orchestratevertebrate brain development.
INTRODUCTION
TheWnt family of secreted lipoproteins controls animal develop-
ment including axial patterning and cell fate specification and
governs tissue homeostasis and stem cell renewal (Clevers
and Nusse, 2012; MacDonald et al., 2009). Anomalies in Wnt
signaling cause human diseases including birth defects, cancer,
and osteoporosis (Clevers and Nusse, 2012; MacDonald et al.,
2009). Wnt proteins engage multiple transmembrane receptors,
including the Frizzled (Fz) serpentine receptors and low-density
lipoprotein receptor-related proteins 5 and 6 (LRP5/6), which
induce stabilization of the transcription co-activator b-catenin
(MacDonald and He, 2012). Wnt proteins act locally near the
source of their secretion in many contexts, and they also behave
as morphogens with long range signaling properties (Hausmann
et al., 2007; Strigini and Cohen, 2000; Zecca et al., 1996; but see
Develo
Alexandre et al., 2014). Critical for these versatile signaling prop-
erties is a lipid modification of Wnt proteins referred to as O-pal-
mitoleoylation (Takada et al., 2006; Willert et al., 2003). This form
of O-acylation, which has been best demonstrated for themouse
Wnt3a, conjugates a mono-unsaturated palmitoleic acid onto
the hydroxyl group of a conserved serine residue (serine 209 of
Wnt3a), likely through the action of a Wnt-specific O-acyltrans-
ferase called Porcupine in the ER (Rios-Esteves et al., 2014; Ta-
kada et al., 2006). Wnt palmitoleoylation serves two essential
functions. First, palmitoleoylation appears to be obligatory for
Wnt secretion, as the Wnt3a(S209A) mutant, which has serine
209 substituted by an alanine, and thus lacks palmitoleoylation,
is not secreted (Takada et al., 2006) possibly as a result of failure
to bind to Wntless, a Wnt chaperone in the secretory pathway
(Coombs et al., 2010; Herr and Basler, 2012; Tang et al., 2012).
Second, the lipid modification is required for secreted Wnt li-
gands to signal, as the palmitoleoylate adduct inserts into a hy-
drophobic cleft of the Fz receptor to form one of the two Wnt-Fz
binding interfaces (Janda et al., 2012).
Canonical Wnt signaling plays multiple roles including axial
patterning and germ layer specification in vertebrate embryo-
genesis (De Robertis and Kuroda, 2004; Hikasa and Sokol,
2013; Stern, 2005). In Xenopus embryos, maternal Wnt/b-cate-
nin signaling promotes the dorsal Spemann-Mangold Organizer
and dorso-ventral (DV) axis formation (Harland and Gerhart,
1997). During gastrulation, a gradient of Wnt/b-catenin signaling
occurs along the anterio-posterior (AP) axis, with higher levels
posteriorly (Kiecker and Niehrs, 2001). The Organizer pro-
motes head development via secreting Wnt antagonists such
as secreted frizzled-related proteins (sFRPs) and Dickkopf-1
(Dkk1), which bind to and inhibit Wnt/Fz and LRP6, respectively
(Cruciat and Niehrs, 2013; De Robertis and Kuroda, 2004). We
recently identified another Organizer-specific and membrane-
tethered Wnt antagonist, Tiki, which is a prototypic Wnt inacti-
vating protease and is required for head formation (Zhang
et al., 2012). The Organizer is also essential for neural induction.
This has been primarily attributed to Organizer-secreted bone
morphogenetic protein (BMP) antagonists such as Chordin and
Noggin, which shield the naive ectoderm from the influence of
BMPs that promote epidermal differentiation, thereby permitting
‘‘default’’ neuralization (De Robertis and Kuroda, 2004; Ozair
et al., 2013; Stern, 2005). Evidence suggests that inhibition of
Wnt signaling and active fibroblast growth factor (FGF) signaling
pmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier Inc. 719
are also required for neural induction in Xenopus and chick em-
bryos (Delaune et al., 2005; Fuentealba et al., 2007; Heeg-Trues-
dell and LaBonne, 2006; Kengaku and Okamoto, 1995; Lamb
and Harland, 1995; Marchal et al., 2009; Pera et al., 2003; Stern,
2005; Wilson et al., 2001). But how regulation of Wnt signaling is
achieved and contributes to neural induction by theOrganizer re-
mains unknown.
Notum (or Wingful) is a secreted antagonist of Wingless (Wg,
Drosophila Wnt1) (Gerlitz and Basler, 2002; Giraldez et al.,
2002). Sequence analysis places Notum in the so-called a/b hy-
drolase superfamily that includes various hydrolytic enzymes
(Nardini and Dijkstra, 1999). Notum appears to regulate Wg
extracellular distribution during wing development (Gerlitz and
Basler, 2002; Giraldez et al., 2002). As heparan sulfate proteogly-
cans, in particular glypicans Dally and Dally-like protein (Dlp), are
involved inmodulating theWg gradient (Han et al., 2005; Yan and
Lin, 2009), and as Notum is in sequence most similar to plant
pectin acetylesterases, Notum was first proposed to modify
glycosaminoglycans (long unbranched polysaccharides) in Dally
and Dlp (Giraldez et al., 2002). Subsequent experiments led to a
revised model that Notum cleaves the glycosylphosphatidylino-
sitol (GPI) anchor of Dlp, thereby switching Dlp from a mem-
brane-anchored activator into a secreted antagonist (Kreuger
et al., 2004). But because Dlp and Dally participate in functions
of most or all morphogen families and play both positive and
negative roles (Beckett et al., 2008; Filmus et al., 2008; Yan
and Lin, 2009), the model that Notum antagonizes Wg signaling
via modifying Dlp is controversial. Notum is conserved from in-
vertebrates to human (Giraldez et al., 2002). In planarians, Notum
and Wnt govern head and tail regeneration, respectively (Pe-
tersen and Reddien, 2011), reflecting a common theme of Wnt
antagonists versus Wnt in AP patterning. In zebrafish, Notum
has a role in DV patterning of the neural tube (Flowers et al.,
2012). But the roles of Notum in early vertebrate embryogenesis
are unknown. Here, we report that Notum is a Wnt-inactivating
deacylase and is critical for neural and head induction by acting
within the presumptive neuroectoderm.
RESULTS
Notum Is a Specific Wnt Antagonist in VertebrateEmbryos and Mammalian CellsThe mouse or human genome harbors a single Notum gene.
Expression of the mouse Notum in HEK293T cells produced
secreted Notum in the conditioned medium (CM) (see below),
and inhibited a prototypic Wnt-responsive TOPFLASH reporter
induced by either co-expression of mouse Wnt3a or treatment
with recombinant human WNT3A proteins (Figures 1A and 1B).
Thus Notum inhibits Wnt3a extracellularly. Notum shares with
other a/b hydrolase proteins a conserved motif GxSxG, in which
the serine mediates the hydrolytic reaction (Nardini and Dijkstra,
1999). Notum(S239A), in which the serine was replaced by an
alanine, was unable to inhibit Wnt3a signaling (Figure 1C),
consistent with results that an analogous Drosophila Notum
mutant fails to inhibit Wg (Giraldez et al., 2002). Thus the enzy-
matic activity of Notum appears to be obligatory for Wg/Wnt
antagonism.
Notum inhibited Wnt signaling in Xenopus laevis embryos.
Ventral injection of Xenopus Wnt8 or b-catenin mRNA into
720 Developmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier
4-cell embryos resulted in axis duplication, and co-injection of
mouse Notum mRNA, but not Notum(S239A) mRNA, inhibited
axis duplication by Wnt8 (Figure 1D). Notum did not inhibit axis
duplication by b-catenin (Figure 1D), consistent with Notum
acting extracellularly. In animal cap explants, Notum inhibited
Wnt8, but not b-catenin, induced expression of Xnr3, a b-catenin
target gene and dorsal marker (Figures 1E and 1F). Importantly,
Notum showed no effect on induction of Xbra, a mesodermal
marker, by Nodal/Xnr1 (a transforming growth factor-bmember)
or basic FGF (bFGF), or induction of Vent2, a ventral marker, by
BMP4 (Figures 1G–1I). These data demonstrate that Notum is a
specific Wnt antagonist, consistent with phenotypic analyses in
invertebrates (Gerlitz and Basler, 2002; Giraldez et al., 2002; Pe-
tersen and Reddien, 2011) and zebrafish (Flowers et al., 2012).
Notum Modifies and Inactivates Wnt ProteinsNotum has been suggested to cleave the GPI anchor of Dlp in
flies (Kreuger et al., 2004). Of the six glypicans encoded in the
mouse/human genome, Glypican 3 (GPC3) and GPC4 have
been implicated in Wnt pathways (Capurro et al., 2014; Filmus
et al., 2008; Sakane et al., 2012). However, in HEK293T cells,
the mouse Notum exhibited minimal GPI-cleaving activity on
GPC3 or GPC4 when compared to a positive control, phospha-
tidylinositol-specific phospholipase C (PI-PLC), which cleaved
the GPI of both glypicans, shedding them into CM (Figures
S1A and S1B).
We previously identified Tiki as a prototypic Wnt modifying
and inactivating enzyme (Zhang et al., 2012). While the Wnt3a
protein is hydrophobic because of its lipid modification (Willert
et al., 2003), Wnt3a modified by Tiki is hydrophilic, as we
demonstrated in a Triton X-114 detergent-aqueous phase sep-
aration assay (Zhang et al., 2012). This observation led to the
suspicion that Tiki might have Wnt deacylase activity, but this
was ruled out by metabolic labeling showing that Wnt3a palmi-
toleoylation was unaltered by Tiki (Zhang et al., 2012). Notum
shares the distinction with Tiki as the only established Wnt
antagonists with enzymatic motifs, raising the possibility that
Notum may modify Wnt proteins. Indeed, Wnt3a when co-ex-
pressed with Notum, like when co-expressed with Tiki, became
predominantly hydrophilic as demonstrated by the Triton X-114
phase separation assay (Figure 2A). Correlating with the in-
ability to inhibit Wnt3a signaling (Figure 1C), Notum(S239A)
did not alter Wnt3a hydrophobicity (Figure 2A). None of several
other secreted or secretory pathway enzymes that exhibit de-
palmitoylation activities including palmitoyl protein thioes-
terases (Zeidman et al., 2009), or are genetically implicated in
Wnt-regulated events such as secreted phospholipase A2
(Cormier et al., 1997), was able to alter Wnt3a hydrophobicity
(data not shown). Therefore, the ability of Notum to modify
Wnt3a hydrophobicity, like that of Tiki, is specific and depen-
dent on its hydrolase motif.
Wnt-induced phosphorylation of the LRP6 co-receptor and
Dishevelled (Dvl), a component downstreamof Fz, indicates acti-
vation of transmembrane signaling (MacDonald and He, 2012).
Wnt3a CM from control HEK293T cells or cells expressing No-
tum(S239A) induced phosphorylation of LRP6, Dvl2, and the
TOPFLASH reporter, but Wnt3a CM from cells that co-ex-
pressed Notum failed to do so (Figures 2B and 2C), implying
Wnt3a inactivation by Notum. But under this condition, secreted
Inc.
Figure 1. NotumAntagonizesWnt Signaling
in Mammalian Cells and Xenopus Embryos
(A) Notum inhibited TOPFLASH induced byWnt3a
in HEK293T cells. Increasing doses of a Notum
expression vector were co-transfected with an
expression vector for Wnt3a.
(B) Notum inhibited TOPFLASH induced by the
recombinant WNT3A protein. HEK293T cells
transfected with increasing doses of the Notum
expression vector were incubated with the
WNT3A protein.
(C) Notum(S239A) lacked the ability to inhibit
TOPFLASH induced by the WNT3A protein.
(D) Notum inhibited axis duplication induced by
Xenopus Wnt8, but not b-catenin, while No-
tum(S239A) did not inhibit axis duplication
induced by either Wnt8 or b-catenin. n, embryos
examined.
(E–I) Notum in animal pole explants inhibited
expression of Xnr3 induced byWnt8 (E), but not by
b-catenin (F), nor did it inhibit Xbra expression
induced by Nodal/Xnr1 (G), or bFGF (H), or Vent2
expression induced by BMP4 (I). EF1a, a loading
control; uninjected embryos, uninj.; whole em-
bryos, WE; whole embryos without the reverse
transcriptase, -RT. There were two doses of
Notum mRNA that were injected.
Error bars (A–C) represent SD of triplicated ex-
periments.
See also Figure S1.
Notum and Wnt3a proteins co-existed in the CM (Figure 2B).
To this end, we generated Notum-TM, which fuses Notum to
a transmembrane domain at the carboxyl terminus and is
anchored on the plasma membrane. Notum-TM was undetect-
able in CM (Figure 2B), but diminished Wnt3a hydrophobicity
and inhibited Wnt3a-induced TOPFLASH as Notum did (Figures
2A and S2A). Wnt3a CM from cells co-expressing Notum-TM, in
contrast to Wnt3a CM from cells co-expressing Notum(S239A)-
TM, induced neither phosphorylation events nor TOPFLASH
(Figures 2B and 2C), suggesting thatWnt3a had been inactivated
by Notum or Notum-TM. Wnt3a from Notum-expressing cells
exhibited poor binding to Fz, as examined using pull-downs
by Fz8 extracellular cysteine-rich domain fused with IgG-Fc
(Fz8CRD-Fc), whereas Wnt3a from Notum(S239A) cells showed
normal Fz binding (Figure 2D). Therefore, Wnt3a modified by
Developmental Cell 32, 719–73
Notum, like that modified by Tiki (Zhang
et al., 2012), is not competent to bind to
the Wnt receptor.
Notum Causes WntDepalmitoleoylationNotum, like Tiki, had no appreciable ef-
fect on Wnt3a secretion, but resulted in
the Wnt3a protein exhibiting faster
mobility during gel electrophoresis (Fig-
ure 2B) (Zhang et al., 2012), consistent
with Wnt3a being modified by Notum.
Tiki cleavage of eight residues at Wnt3a
amino terminus causes the change in
Wnt3a hydrophobicity and electropho-
retic mobility (Zhang et al., 2012). However, unlike Tiki, Notum
did not exhibit amino terminal cleavage activity toward Wnt3a
(Figure S2B). Thus, despite the fact that Wnt3a modified by
Notum and Tiki behaved similarly, Notum is unlikely to be a
Wnt protease, and therefore modifies Wnt3a in a fundamentally
different manner.
Mass spectrometry of Wnt3a and the Wnt8 crystal structure
suggest that O-palmitoleoylation and N-glycosylation are the
predominant modifications of an active Wnt protein (Janda
et al., 2012; Takada et al., 2006; Zhang et al., 2012). Others
and we have shown that glycosylation/de-glycosylation does
not alter Wnt3a hydrophobicity (Komekado et al., 2007; Zhang
et al., 2012). These considerations raise the possibility that
Notum may be a Wnt deacylase that removes palmitoleic acid
through hydrolysis, thereby inactivating Wnt. We performed
0, March 23, 2015 ª2015 Elsevier Inc. 721
Figure 2. Notum Causes Wnt Deacylation
and Inactivation
(A) The Wnt3a protein modified by Notum became
hydrophilic in the Triton X-114 detergent-aqueous
phase separation assay. Wnt3a from mock or
Notum(S239A)-expressing cells was hydrophobic
and partitioned in the detergent (De) phase, but
Wnt3a from Notum- or Notum-TM-expressing
cells partitioned in the aqueous (Aq) phase; total
input, T.
(B) Wnt3a CM from Notum- or Notum-TM-ex-
pressing cells was inactive and induced minimal
phosphorylation of LRP6 and Dvl2 in mouse
embryonic fibroblast cells, whereas Wnt3a CM
from mock, Notum(S239A)-, or Notum(S239A)-
TM-expressing cells induced phosphorylation
of LRP6 and Dvl2. Note that Wnt3a from Notum-
or Notum-TM-expressing cells exhibited slightly
faster migration. WCL, whole cell lysates.
(C) Wnt3a CM from Notum or Notum-TM-ex-
pressing cells induced minimal TOPFLASH in
HEK293T cells, whereas Wnt3a CM from mock,
Notum(S239A)-, or Notum(S239A)-TM-expressing
cells induced TOPFLASH. Error bars represent SD
of triplicated experiments.
(D) Wnt3a secreted from Notum-expressing cells
exhibited minimal binding to mFz8CRD-IgG,
whereas Wnt3a secreted from mock or No-
tum(S239A)-expressing cells exhibited binding.
(E) Notum, but not Notum(S239A), reduced Wnt3a
acylation when they were co-expressed in
HEK293T cells.
See also Figure S2.
metabolic labeling of Wnt3a with a palmitic acid analog (az-15)
(Zhang et al., 2012), which can be desaturated and incorporated
into Wnt3a in the cell (Hannoush, 2012; Rios-Esteves and Resh,
2013; Zhang et al., 2012). The resulting lipidation of Wnt3a can
be detected using a fluorescent dye through click chemistry
(Zhang et al., 2012). Indeed, Wnt3a CM from control or No-
tum(S239A)-expressing cells exhibited strong lipid labeling indi-
cating palmitoleoylation, but Wnt3a CM from Notum-expressing
cells lacked lipid labeling (Figure 2E). Therefore, Notum through
its hydrolase motif causes Wnt3a depalmitoleoylation.
Notum Is Likely a Wnt Deacylase and ActsExtracellularlyWnt palmitoleoylation appears to be essential for Wnt secretion,
as Wnt3a(S209A), which lacks the palmitoleoylated serine, is not
secreted, nor are any of the Wnt proteins that are expressed in
cells/embryos deficient for Porcupine (Barrott et al., 2011; Bie-
722 Developmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier Inc.
chele et al., 2011; Proffitt and Virshup,
2012; Takada et al., 2006). Notum does
not affect Wnt3a secretion (Figure 2B),
suggesting that depalmitoleoylation oc-
curs after Wnt3a secretion. Indeed, in
contrast to secreted Wnt3a (Figure 2A),
hydrophobicity of Wnt3a from cell lysates
was less affected by Notum (Figure 3A),
suggesting that Wnt3a in the secretory
pathway is insensitive to Notum. Impor-
tantly, incubation of recombinantWNT3A in NotumCM in culture
resulted in loss ofWnt3a hydrophobicity (Figure 3B). This result is
consistent with Notum inhibition of recombinant WNT3A pro-
teins added to responding cells (Figure 1B). We purified, from
respective CM, secreted Notum, Notum(S239A), and Wnt3a
that had been metabolically labeled by the palmitic acid analog
and reconstituted the depalmitoleoylation reaction in vitro.
Notum, but not Notum(S239A), caused Wnt3a deacylation (Fig-
ure 3C). The simplest interpretation of these results is that Notum
is a Wnt deacylase.
Palmitoleoylation Is Required for the Wnt Structure andActive Monomeric StateWnt3a cleaved by Tiki retains normal palmitoleoylation, but is hy-
drophilic (Zhang et al., 2012). This paradox appears to be ex-
plained by our finding that Tiki-cleaved Wnt3a (or an engineered
Wnt3aDN that lacks the eight amino terminal residues removed
Figure 3. Notum Is Likely a Wnt Deacylase Acting Extracellularly and Causes Wnt3a and Wnt5a to Form Oxidized Oligomers
(A) Notum had minimal effects on hydrophobicity of Wnt3a from WCL in the detergent-aqueous phase separation assay.
(B) Recombinant WNT3A proteins lost hydrophobicity after incubation with Notum-expressing cells.
(C) Notum reduced Wnt3a acylation in vitro. Purified metabolically labeled Wnt3a protein was incubated with mock, purified Notum, or Notum(S239A)
proteins.
(D) Wnt3a secreted from Notum-, but not Notum(S239A)-, expressing cells formed oxidized oligomer. Wnt3a CM from mock, Notum-, or Notum(S239A)-ex-
pressing cells was analyzed by non-reducing or reducing SDS-PAGE. Wnt3a monomers and oxidized oligomers were labeled by an arrow and asterisk,
respectively.
(E) Recombinant WNT3A proteins formed oxidized oligomers upon incubation with Notum-, but not mock or Notum(S239A)-,expressing cells.
(F) Wnt5a proteins secreted from Notum-expressing cells formed oxidized oligomers.
See also Figure S2.
by Tiki) forms large, but soluble, Wnt3a oligomers that are linked
by inter-Wnt3a disulfide bonds (Zhang et al., 2012). These
oxidizedWnt3a oligomers behave in a strictly hydrophilicmanner
in the Triton X-114 phase separation assay, and thus may have
buried the lipid adduct inside the oligomer (Zhang et al., 2012).
It is worth noting that Wnt3a amino terminal residues cleaved
by Tiki do not contain a cysteine, yet their absence profoundly
alters Wnt3a structural integrity and disulfide bond patterns
(Zhang et al., 2012). Unexpectedly, Wnt3a modified by Notum
behaved in a similar manner, it formed soluble and large Wnt3a
oligomers that were linked by inter-Wnt3a disulfide bonds and
detected under non-reducing conditions (Figure 3D). Wnt3a
CM from control or Notum(S239A)-expressing cells yielded
mostly monomeric proteins under the same non-reducing condi-
tion (Figure 3D). This conversion of the active/monomeric form
Develo
into inactive/oxidized oligomeric formsbyNotumoccurred extra-
cellularly, because recombinant WNT3A, which is active/mono-
meric, became oxidized oligomers upon incubation in vitro with
Notum, but not with Notum(S239A), CM (Figure 3E). Such a con-
version after depalmitoleoylation by Notum was not a unique
property of Wnt3a, Wnt5a appeared to be deacylated and lose
its hydrophobicity upon co-expression with Notum (Figure S2C)
and was converted concomitantly from monomers to oxidized
oligomers (Figure 3F). Therefore, palmitoleoylation is essential
for the Wnt structure that ensures its active monomeric state.
Notum Is Expressed Dynamically during Xenopus
EmbryogenesisNotum functions in vertebrates have been studied in zebrafish,
which have three Notum genes, Notum1a, Notum1b, and
pmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier Inc. 723
Figure 4. NotumExpression Patterns during
Xenopus Embryogenesis
(A and B) RT-PCR revealed that Notum is mater-
nally and zygotically expressed throughout Xen-
opus embryogenesis (A), and is enriched animally
and dorsally at stage 10.5; animal caps, AC; dorsal
marginal zone, DMZ; ventral marginal zone, VMZ;
vegetal caps, VC; a pan-mesodermal marker,
Xbra; a dorsal marker, Chordin; an animal and
ventral marker, Msx1.
(C–T) Whole mount in situ hybridization for Notum/
Notum’ expression, showing lateral view at 4-cell
and stage 6.5 (C and D); lateral and bisected view
at stage 8.5 (E and F); bisected view at stage 9.5
(G); lateral and bisected view at stage 10.5, with
dorsal on right (H and I); dorsal and bisected view
at the stage 11 (J and K); with an enlarged anterior
view showing stronger expression anteriorly (L);
dorso-anterior (M); dorsal (N, anterior on top); and
cross-section (O, dorsal on top; and P, an enlarged
view) at stage 15; lateral view at the tail bud stage
(Q); with an enlarged anterior view indicating
expression in the cement gland (R, arrow); lateral
view at the early tadpole stage (S); and with an
enlarged view indicating expression in the pro-
nephros region (T, arrowhead). The blue color at
the blastocoel surface in bisections was non-
specific.
See also Figure S3.
Notum2 (Cantu et al., 2013; Flowers et al., 2012). Notum1a and
1b are paralogs due to teleost genome duplication and are
orthologs of the mammalian/Drosophila Notum, and indeed,
Notum1a antagonizes Wnt/b-catenin signaling (Flowers et al.,
2012). Depletion of Notum1a causes neural tube defects in DV
patterning (Flowers et al., 2012). Notum2 is a distant relative of
mammalian/Drosophila Notum and does not appear to have
Wnt antagonist activity (Cantu et al., 2013).Notum2 is strictly ex-
pressed in muscle pioneer cells in larvae and has a role in axon
guidance during primary motor neurogenesis (Cantu et al.,
2013), which is unique to teleosts and amphibians (to endow
larvae with escape responses from predators, for example).
The Xenopus laevis (allotetraploid) genome contains Notum
and Notum’, which are pseudo-alleles due to genome duplica-
tion, and Notum2 (Figure S3). Notum and Notum’ are 94% iden-
tical in nucleotide (and corresponding amino acid) sequences
and are orthologs of the mammalian/Drosophila Notum and
fish Notum1a, whereas Notum2 is the ortholog of fish Notum2
(Figure S3). The Xenopus tropicalis (diploid) genome contains a
single Notum gene, and Notum2 (Figure S3).
Notum and Notum’ mRNAs are expressed in the egg (and
are enriched in the animal half, data not shown) and through
cleavage to gastrulation stages (Figure 4A) and are enriched
in the animal (prospective ectoderm) and dorsal regions
in early gastrula (Figure 4B). Whole mount in situ hybridiza-
tion showed expression of Notum and Notum’ (as the two
pseudo-alleles were likely cross-hybridized by the probe) in an-
724 Developmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier Inc.
imal blastomeres at 4-cell and stage 6.5
(Figures 4C and 4D). At stages 8.5 and
9.5 (blastula), Notum mRNA was de-
tected broadly in the animal region
(Figures 4E–4G). At stage 10 (early gastrula), Notum mRNA re-
mains broadly expressed animally, but was also detected in
the dorsal marginal zone (the Organizer), with lower expression
in the ventral marginal zone (Figures 4H and 4I). At stage
11, Notum mRNA was found in the forming neural plate in a
noticeable AP gradient (anterior high and posterior low), with
additional weaker expression in the head mesoderm (Figures
4J–4L). Notum mRNA remains detectable, but becomes faint
in the neural plate at stage 13 (data not shown). By stage 15,
Notum mRNA was detected at the anterior border of the neural
plate and in ventro-lateral epidermis excluding the neural plate
(Figures 4M and 4N). Cross-section of a stage 15 embryo
showed Notum expression in the lateral surface of the
epidermis and the lateral plate mesoderm (Figures 4O and
4P). Later, Notum was detected in the cement gland (an ante-
rior organ) at tail bud stages (stage 25, Figures 4Q and 4R), in
branchial arches, the otic vesicle, and developing pronephros,
with diffused expression in the head (stage 35, Figures 4S and
4T). Thus, Notum mRNA exhibits dynamic expression during
Xenopus embryogenesis, in particular during neural induction
and AP patterning.
Notum Is Required for Head FormationXenopus Notum and Notum’ behaved identically as the mouse
Notum in Wnt deacylation and inactivation (Figure S4). Dorsal
injection of synthetic mRNA for the mouse Notum, but not
Notum(S239A), induced an enlarged head as seen for Tiki1 or
Figure 5. Notum Is Required for Anterior Development in Xenopus Embryos(A and B) Dorsal injection of Notum mRNA, but not Notum(S239A), induced an enlarged head similar to that induced by Tiki1 mRNA, and resulting phenotypes
were tabulated.
(C) The NotumMO and the Notum’MO inhibited protein synthesis from Xenopus Notum (xNotum) or Notum’ (xNotum’), but not mouse Notum (mNotum) mRNA.
Co, MO, and MO’ indicate control MO and MOs against xNotum and xNotum’, respectively.
(D and E) Dorso-animal injection of the two NotumMOs together caused anterior defects that were rescued bymNotum mRNA, and resulting phenotypes were
tabulated.
(F) The Notum MOs suppressed expression of forebrain markers Otx2 and Bf1 and a hindbrain marker Krox20 at stage 17. See also Table S1.
(G) Illustration of the MO-injected A1 and B1 blastomeres at 32-cell stage and their descendent tissues at stage 10.5.
(H) The Notum MOs injected into the A1 blastomeres caused anterior defects in more embryos (69%) than those injected into the B1 blastomeres (22%).
See also Figures S4 and S5; Tables S2 and S3.
Dkk1 mRNA injection (Figures 5A and 5B) (Zhang et al., 2012),
consistent withWnt inhibition in embryos.We designed twomor-
pholino antisense oligonucleotides (MOs) against the 50 regionsurrounding the ATG initiation codon of Notum and Notum’.
These two MOs blocked protein synthesis from mRNAs for Xen-
opus Notum andNotum’, respectively, but not themouseNotum
(Figure 5C) that does not have the MO-targeting sequence,
demonstrating the specificity of these MOs. Dorso-animal injec-
tion of the two MOs together, but not of a control MO, at the
8-cell stage caused severe deficiency in head formation, result-
ing in embryos lacking the forebrain, eyes, and the cement gland
(Figures 5D and 5E). These anterior defects were rescued when
theMOs were co-injected with mouseNotummRNA (Figures 5C
Develo
and 5E), illustrating that the phenotypes were a result of lacking
Notum. These anterior defects by the Notum MOs were also
rescued/over-rescued by co-injection of Dkk1 mRNA (Fig-
ure S5A) and by co-injection of a b-catenin MO that depletes
the b-catenin protein (Heasman et al., 2000) (Figures S5B and
S5C), supporting the notion that the defects were due to exces-
sive Wnt/b-catenin signaling. The overt anterior deficiencies by
the Notum MOs were accompanied by, at stage 17, diminished
expression of anterior markers Otx2, Bf1, Pax6 (forebrain), and
XAG (the cement gland), and of a hindbrain marker Krox20,
each of which was rescued by co-injection of the mouse Notum,
but notNotum(S239A), mRNA (Figures 5F, S5D, and S5E; Tables
S1 and S2). The expression of the spinal cord markerHoxb9was
pmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier Inc. 725
unaffected by the Notum MOs (Figure S5E). Thus, like Tiki1,
Notum is required for Xenopus head patterning.
Notum Functions in Prospective EctodermTiki1 is specifically expressed in the Organizer, in particular, the
so-called head organizer that is composed of endomesodermal
tissues underlying the future forebrain (Zhang et al., 2012). In
Tiki1-depleted embryos, the head organizer function is signifi-
cantly compromised, as evidenced by diminished expression
of all head organizer markers examined, including Dkk1, Chor-
din, Lim1, Goosecoid, and Otx2, while the expression of other
dorsal markers such as Xnr3 and Xnot1 is unaffected (Zhang
et al., 2012). However, in Notum-depleted embryos, the expres-
sion of head organizer markers Chordin andGoosecoid, like that
of Xnr3 and Xnot1, was unchanged (Figure S5F; Table S3). Thus,
unlike Tiki1, Notum function appears to minimally impact the
head organizer integrity and may therefore participate in head
patterning by acting within the prospective ectoderm, in which
Notum is expressed during blastula and gastrula stages (Figures
4E–4L).
To examine this issue further, we injected, at the 32-cell stage,
Notum MOs into the dorsal A1 blastomeres, which give rise
mostly to the future head and neuro-ectoderm, or the dorsal
B1 blastomeres, which are fated primarily to become the dorsal
Organizer (Dale and Slack, 1987; Moody, 1987) (Figure 5G).
Notum depletion in A1 and B1 progenies showed drastically
different outcomes and resulted in head deficiencies in 69%
and 22% of embryos, respectively (Figure 5H), supporting the
notion that Notum is required primarily in the prospective ecto-
derm for anterior neural development.
Notum Is Required for Neural InductionInhibition of Wnt signaling has been suggested to be critical for
neural induction in Xenopus (Fuentealba et al., 2007; Heeg-
Truesdell and LaBonne, 2006), but the profound effect of Wnt
signaling on dorsal Organizer, which induces neural tissues
through secreting BMP antagonists, complicates studies on
this issue (Stern, 2005). Interestingly, embryos injected with
the Notum MOs, but not the control MO, showed a significant
reduction of a pan-neural marker Sox2, but an expansion of
the epidermal marker cytokeratin, and these reciprocal changes
were rescued by co-injection of the mouse Notum, but not
Notum(S239A), mRNA (Figure 6A; Table S4). Because Organizer
markers were unaffected (Figure S5E), these results suggest
that Notum acts within prospective ectoderm for neural
specification.
To examine this issue directly, we employed a classical neural
induction assay using animal cap explants. Chordin or Noggin
induced pan and anterior neural markers and suppressed
the epidermal marker in explants from control embryos, but
failed to do so in explants from Notum-depleted embryos, and
this neural induction defect was rescued by the mouse Notum,
but notNotum(S239A), mRNA (Figures 6B andS6A). Importantly,
the ability of Chordin to induce neural tissue was restored when
the b-catenin MO was co-injected with the Notum MOs (Fig-
ure 6C), suggesting the failure of neural induction upon Notum
depletion as a result of excessive Wnt/b-catenin signaling.
To rule out the possibility that Notum is required for the BMP
antagonists to function properly, we showed that Chordin or
726 Developmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier
Noggin inhibited BMP4 induction of the ventral Vent2 regardless
of Notum depletion (Figure S6B). Thus, Notum suppression of
Wnt signaling is a prerequisite for neural induction by BMP an-
tagonists. Indeed, overexpression of Notum, like that of Dkk1
(Fuentealba et al., 2007; Glinka et al., 1998), induced pan and
anterior neural markers in animal explants (Figure 6D).
DISCUSSION
Notum as a Wnt Deacylase: Comparisons with the WntProtease TikiGenetic analyses in planarian and zebrafish show that Notum in-
hibits signaling byWnt, but not byHedgehog or other growth fac-
tors (Flowers et al., 2012; Petersen andReddien, 2011).We show
that the mouse or Xenopus Notum is a Wnt3a antagonist in
mammalian cells and inhibits target gene activation by Wnt8,
butnotBMP,Nodal, or FGF inXenopusanimal explants (Figure1).
Notum belongs to the a/b hydrolase superfamily that includes
peptidases, lipases, esterases, and other hydrolytic enzymes
(Nardini and Dijkstra, 1999). It was proposed that Notum inhibits
Wg signaling via hydrolyzing the GPI anchor of the Dlp glypican
(Kreuger et al., 2004). But this model is strongly challenged by
the fact that Dlp, Dally, and their homologs participate in most
or all morphogen signaling pathways (Filmus et al., 2008).
Compared to PI-PLC, we detected minimal Notum cleavage of
the GPI anchor of GPC3 or GPC4 (Figure S1), two mammalian
glypicans that have been implicated in Wnt pathways (Capurro
et al., 2014; Filmus et al., 2008; Sakane et al., 2012).
Tiki is an archetypal Wnt antagonist that modifies/cleaves the
Wnt ligand (Zhang et al., 2012). Tiki and Notum represent a
unique group of extracellular Wnt inhibitors with catalytic capac-
ities. We show that Notum is also aWnt-inactivating enzyme and
shares several commonalities with Tiki in modifying Wnt sub-
strates (Table 1): neither affectsWnt secretion; and either causes
Wnt to exhibit (1) faster electrophoreticmobility, (2) loss of hydro-
phobicity, (3) oxidized oligomer formation, and importantly, (4)
loss of receptor-binding and signaling activity (Figures 2, 3, S2,
and S4). Despite these similarities and that both Tiki and Notum
are hydrolytic enzymes, the natures of their Wnt-inactivating
modifications are different: Tiki cleaves the amino terminal resi-
dues of Wnt proteins as a protease (Zhang et al., 2012), whereas
Notum cleaves the palmitoleoylate adduct of Wnt proteins as a
deacylase (Figures 2 and 3). Therefore, distinct Wnt-inactivating
enzymes operate in modulating Wnt signaling, with Notum being
secreted and potentially diffusible, whereas Tiki being mem-
brane-tethered and affecting a cell autonomously or nearby
(Zhang et al., 2012). The recently determined structure of Notum
reveals an a/b hydrolase fold with a large hydrophobic cavity,
into which docking of a palmitoleoylate positions the acyl-oxy-
ester bond in proximity to the GxSxG catalytic center, providing
a structural basis for Notum deacylase function (Kakugawa et al.,
2015). Notum deacylates Wnt3a and possibly Wnt5a, but Notum
specificity towardWnt proteins and different Wnt signaling path-
ways remain to be studied.
Wnt Lipidation as a Requirement for Its Structure andActive Monomeric ConformationWnt palmitoleoylation has two critical functions; it may be
recognized by Wntless, a Wnt chaperone in the secretory
Inc.
Figure 6. Notum Is Required for Neural In-
duction in Embryos and Animal Explants
(A) The Notum MOs reduced the expression
domain of Sox2, a pan-neural marker, and ex-
panded the expression domain of cytokeratin, an
epidermal marker, and the reciprocal changes
were rescued by mNotum, but not mNu-
tum(S239A), mRNA. See also Table S4.
(B) The Notum MOs suppressed expression of
neural markers induced by injection of the Chor-
din mRNA and restored cytokeratin expression
that were inhibited by Chordin. The effect of
Notum MOs was rescued by mNotum, but not
mNotum(S239A), mRNA; a cement gland marker,
XAG; anterior neural markers, Bf1 and Pax6;
a pan-neural marker, Sox2; a neuronal marker,
N-tubulin; an epidermal marker, Keratin; a
mesodermal marker, M-Actin; and a loading
control, ODC.
(C) Chordin induced Bf1 and Sox2 expression
when the Notum MOs and a b-catenin MO were
co-injected.
(D) Injection of Notum mRNA, like that of Chordin
mRNA, induced expression of neural markers
Sox2 and Pax6 and suppressed that of an epi-
dermal marker, cytokeratin, in animal cap ex-
plants.
See also Figure S6.
pathway (Coombs et al., 2010; Herr and Basler, 2012; Tang
et al., 2012), and it is essential for Wnt binding to Fz (Janda
et al., 2012). We show that Wnt depalmitoleoylation by
Notum occurs extracellularly, likely accounting for normal
secretion, but lack of receptor binding by deacylated Wnt3a
(Figures 1, 2, and 3). But unexpectedly, Wnt3a and Wnt5a de-
palmitoleoylated by Notum, similar to Wnt3a that is amino-
terminally cleaved by Tiki (Zhang et al., 2012), form large,
yet soluble, Wnt oligomers that are covalently linked by
inter-Wnt disulfide bonds (Figure 3). These oxidized Wnt
oligomers generated by Notum or Tiki appear to be similar
and are recapitulated by the Wnt3aDN mutant that lacks
Developmental Cell 32, 719–73
the amino terminal residues cleaved
by Tiki (Zhang et al., 2012). Therefore,
the Wnt oxidized oligomeric state (or
states), which is prevalent during Wnt
biogenesis (Zhang et al., 2012), may
be a default state of the Wnt protein.
Wnt palmitoleoylation thus appears to
have a third critical role; i.e., for main-
taining Wnt structural integrity to en-
sure its active monomeric conforma-
tion. The intact Wnt amino terminus,
which is rich in hydrophobic residues,
plays a similar role in maintaining the
Wnt fold (Zhang et al., 2012). The struc-
tural basis for the requirement of the
lipid, and of the Wnt amino terminus,
for the Wnt fold is not yet obvious
from the Wnt8 crystal structure (Janda
et al., 2012). Given that lipid modi-
fications of proteins are common in
signaling, a general role for lipid adducts in protein conforma-
tion deserves investigation.
Notum in Vertebrate Neural and Head Induction:Comparisons with the Role of TikiWhile Tiki1 is expressed in the Organizer and its descendent
head organizer during gastrulation (Zhang et al., 2012), Notum
(includingNotum’) ismaternally expressedand isenriched innaive
ectoderm (Figure 4).Overexpression ofNotumor Tiki1 dorsally re-
sults in head enlargement; depletion of Notum or Tiki1 causes
deficiency in head formation (Figure 5) (Zhang et al., 2012).
Thus, these two Wnt-inactivating enzymes are each required for
0, March 23, 2015 ª2015 Elsevier Inc. 727
Table 2. Comparisons of Tiki1 and Notum Expression and
Functions in Xenopus Embryos
Tiki1 Notum
Expression
stages
from early
gastrulation
from egg throughout
embryogenesis
Early expression
patterns
organizer ectoderm, forming neural
plate, and anterior neural
plate border
Depletion
phenotypes
headless headless and neural plate
reduction
Action sites
(tissues)
head organizer ectoderm
Extracellular
features
membrane tethered
(non-diffusible)
secreted (diffusible?)
Table 1. Comparisons of Wnt3a Modifications by Tiki and Notum
Wnt3a Wnt3a + Tiki Wnt3a + Notum
Secretion normal normal normal
Hydrophobicity hydrophobic hydrophilic hydrophilic
Mobility in gel normal faster faster
Oxidized oligomer no yes yes
Signaling activity yes no no
Binding to receptor yes no no
Nature of
modification
� N-terminal
cleavage
deacylation
anterior development and not redundant. The action sites of
Notum and Tiki1 are distinct and complementary (Table 2). Tiki1
acts within the head organizer (Zhang et al., 2012), which induces
overlying ectoderm to form anterior neural tissues, whereas
Notum primarily acts within ectoderm to endow it with compe-
tence to become anterior brain, and Notum depletion does not
affect Organizer formation (Figures 5 and S5; Table 2).
The action of Notum within ectoderm appears to explain its
significant, but unique, role among Wnt antagonists in neural in-
duction. Upon Notum depletion, the expression of anterior and
pan neural markers is reduced, while that of an epidermal marker
is expanded (Figure 6), suggesting neural to epidermal fate con-
version. Naive ectoderm with Notum depletion loses its compe-
tence to respond to neural inducers/BMP antagonists (Figures 6
and S6). Conversely, overexpression of Notum, like that of Dkk1
(Fuentealba et al., 2007; Glinka et al., 1998), induces anterior
neural tissues (Figure 6). Thus, Wnt inactivation by Notum within
ectoderm is a critical part of the ‘‘default’’ neural fate (De Rob-
ertis and Kuroda, 2004; Ozair et al., 2013; Stern, 2005). This
requirement of Wnt inhibition by Notum, in addition to that of
BMP inhibition (and of FGF activation), for neuralization in Xeno-
pus seems to mirror neural induction mechanisms described in
the chick (Stern, 2005; Streit et al., 1998, 2000; Wilson et al.,
2000, 2001), supporting a unifying model for neural fate determi-
nation in vertebrates. The dependence onWnt and BMP co-inhi-
bition (and FGF activation) for neural induction may act through
parallel signaling pathways and/or through crosstalks by these
pathways (Fuentealba et al., 2007; Pera et al., 2003).
Notum exhibits dynamic expression in naive ectoderm in blas-
tula, in the forming neural plate in early gastrula, and at the ante-
rior border of (and exclusion from) the neural plate in late gastrula
(Figure 4). Interestingly, its ectodermal expression encompasses
the so-called blastula Chordin and Noggin expression (BCNE)
center, which represents the dorsal ectoderm giving rise to
future fore-, mid-, and hindbrain (Kuroda et al., 2004). The tran-
sient ectodermal Chordin and Noggin expression in blastula is
required for dorsal ectoderm to become anterior neural tissues
that are reinforced by Chordin and Noggin from the Organizer
during gastrulation (Kuroda et al., 2004). Similarly, we propose
that ectodermal Notum expression in blastula primes the dor-
sal ectoderm for sustained neuralization during gastrulation by
Organizer-generated BMP and Wnt antagonists, thereby under-
lying its requirement for neural and anterior induction. Subse-
quent Notum expression in the anterior neural plate border
may further reinforce Wnt inactivation for patterning forebrain
and surrounding regions such as pre-placodal ectoderm, which
728 Developmental Cell 32, 719–730, March 23, 2015 ª2015 Elsevier
is induced to form placodes through Wnt inhibition (Litsiou et al.,
2005). Experiments will be required to dissect the temporal
order and potentiallymultiple roles of Notumduring neural induc-
tion and patterning. Our results demonstrate that distinct Wnt
inactivation mechanisms by Notum in naive ectoderm and Tiki
in Organizer coordinate early brain formation, and that Notum
function in head development is conserved from planarians to
vertebrates. Notum as a Wnt inactivating enzyme represents a
potential therapeutic target.
EXPERIMENTAL PROCEDURES
Plasmids
Themouse Notum cDNA in pCAGGS contains the full coding region fusedwith
a33FLAG tagandwas fromDrs. S. Park andS. Sockanathan.GPC3andGPC4
expressing vectors were from A. Kikuchi. Notum coding region fused with a
13FLAG tag was subcloned into pCS2+. Notum(S239A) was generated by
PCR-based mutagenesis method based on pCS2+/Notum-FLAG. Notum-TM
andNotum(S239A)-TMwere constructedby inserting a cDNA fragment encod-
ing the transmembrane domain of epidermal growth factor receptor (EGFR)
(IATGMVGALLLLLVVALGIGLFM) between Notum and FLAG tag. Xenopus
laevis Notum and Notum’ cDNAs were amplified from embryo cDNAs via RT-
PCR and cloned into pCS2+. Details of plasmids are available upon request.
Triton X-114 Phase Separation
Triton X-114 phase separation was performed as described (Zhang et al.,
2012). Briefly, for CM separation, Wnt3a or Wnt5a CM was mixed with equal
volume of Triton X-114 buffer (10 millimolar (mM) Tris-HCl [pH 7.4], 150 mM
NaCl, and 4.5% Triton X-114); for whole cell lysate (WCL) separation, WCL
containing 1% Triton X-114 was mixed with equal volume of 3.5% Triton X-
114 buffer. The mixtures were incubated on ice for 5 min and then at 37�Cfor 5 min. After centrifugation at 2,000 g for 5 min, the top aqueous phase,
the bottom detergent phase, and the original mixture (total) were analyzed
by SDS-PAGE and western blotting.
Metabolic Labeling and Click-Chemistry
Metabolic labeling and click-chemistry assays were performed as described
(Zhang et al., 2012). Briefly, HEK293T cells or HEK293T cells stably expressing
hemagglutinin (HA)-Wnt3a (Zhang et al., 2012) in 60 millimeters (mm) tissue
culture dishes were transfected with indicated plasmids. At 24 hr after trans-
fection, cells were washed with serum-free DMEM once and then cultured in
the labeling medium (DMEM containing 5% dialyzed fetal bovine serum
[FBS], 40 mM az-15, or alk-14) for 12 hr. CM was collected and cleared by
centrifugation. HA-Wnt3a was enriched fromCMby anti-HA beads. The beads
were washed and re-suspended in 20 ml of PBS plus 2.25 ml freshly premixed
click-chemistry reaction mixture (alk-Rho or az-Rho, 100 mM, Tris(2-carbox-
yethyl)phosphine [TCEP], 1 mM, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]
Inc.
amine [TBTA], 100 mM, and CuSO4$5H2O, 1mM) for 1 hr at room temperature.
The samples were denatured and separated by SDS-PAGE, and the gels were
fixed and scanned on a GE Healthcare Typhoon 9400 variable-mode imager
for Rhodamine-associated signal at excitation 532 nanometers (nm)/emission
580 nm. The same samples were also subjected to SDS-PAGE and western
blotting to detect total Wnt3a protein.
Protein Purification and In Vitro Deacylation of Wnt3a
The CM from HEK293T cells expressing Notum-FLAG or Notum(S239A)-
FLAG was mixed with anti-FLAG beads and incubated at 4�C overnight.
The beads were washed with PBS/0.1% Triton X-100 and the FLAG fusion
proteins were eluted with elution buffer (50 mM HEPES (pH 7.4), 100 mM
NaCl, 0.1% Triton X-100, and 50 mg/ml 33FLAG peptide). For in vitro deacy-
lation, alk-14 labeled Wnt3a in CM was immunoprecipitated with anti-HA
agarose beads and incubated with purified Notum or Notum(S239A) protein
at room temperature overnight, followed with click-chemistry reaction. The
reaction mixtures were denatured and separated by SDS-PAGE. The fluores-
cent signal and total Wnt3a proteins were detected by in-gel scanning and
western blotting.
Xenopus Embryo Manipulations
Frogs (Xenopus laevis) were maintained at the Animal Resource Children’s
Hospital (ARCH) in accordance with institutional guidelines. Procedures for
embryo manipulation, RT-PCR, and in situ hybridization were performed as
previously described (Zhang et al., 2012). The full-length Xenopus laevis
Notum’ cDNA was used to make in situ probe.
mRNA and Morpholino Injection
Axis duplication and animal cap assays were performed as described (Zhang
et al., 2012). For axis duplication assays, Xwnt8 (1 picogram [pg]) or b-catenin
(50 pg) mRNA was injected alone or together with Notum (200 pg) mRNA into
the ventral marginal zone at the 4- or 8-cell stage, and the phenotype was
scored at the tadpole stage. GFP was used as a negative control. For animal
cap assays, indicatedmRNAs (Notum, 100 and 200 pg; Xwnt8, 5 pg; b-catenin
30 pg; Xnr1, 250 pg; BMP4, 100 pg; and Chordin, 200 pg) and morpholinos (20
nanograms [ng]) were injected into the animal pole at the 4-cell stage, and an-
imal caps were dissected at stage 9 (in the case of Xwnt8, Xnr1, BMP4, and
Chordin), or at stage 8.5 and treated with recombinant proteins (bFGF,
100 ng/ml and Noggin, 500 ng/ml) until stage 11 (for Xwnt8, Xnr1, BMP4,
and bFGF) or stage 18 (for Noggin or Chordin) before RT-PCR. To examine
Notum and Notum’ MO specificity, 500 pg of xNotum, xNotum’, or mNotum
mRNAwas injected with control MO,Notum, orNotum’MO (20 ng) into the an-
imal pole at the 2-cell stage and cultured until stage 10 for western blotting. To
knockdown the endogenous xNotum and xNotum’, 20 ng of control MO or
Notum and Notum’ MOs were injected into two dorso-animal blastomeres at
the 8-cell stage, or two dorsal A1 or B1 cells at the 32-cell stage, and the
phenotype was scored at stage 35. To rescue the MOs (xNotum and xNotum’)
phenotype, 200 pg of mNotum, 20 pg of DKK1 mRNA, or 10 ng of b-catenin
MO was injected together with MOs.
ACCESSION NUMBERS
The GenBank accession numbers for the Xenopus laevis Notum, Notum’, and
Notum2 reported in this paper are KP781855, KP781856, and KP781857,
respectively.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
six figures, and four tables and can be found with this article online at http://
dx.doi.org/10.1016/j.devcel.2015.02.014.
AUTHOR CONTRIBUTIONS
X.Z. performed all biochemical experiments. S.-M.C. performed most Xeno-
pus experiments. N.G.A., A.H.R., and J.G.A. performed additional Xenopus
experiments. B.T.M. contributed to experiments in mammalian cells and per-
Develo
formed bioinformatic analysis. X.Z., S.-M.C., J.G.A., M.Z., E.Y.J., and X.H.
contributed to experimental designs, X.Z., S.-M.C., and X.H. wrote the manu-
script, and all co-authors edited the manuscript.
ACKNOWLEDGMENTS
We thank S. Park, S. Sockanathan, and A. Kikuchi for plasmids, J.-P. Saint-
Jeannet for comments, and J.-P. Vincent for discussion. X.Z. and S.-M.C.
were in part supported by a grant from Harvard William Randolph Hearst
Fund and a fellowship from National Research Foundation of Korea, respec-
tively. N.G.A. is in part supported by a postdoctoral fellowship from Science
Without Borders program/Council for Research and Technology (CNPq) of
Brazil. M.Z. held a Marie Curie Intra European Fellowship (IEF) fellowship.
J.G.A. and A.H.R. are supported by CNPq and Rio de Janeiro State Founda-
tion for Science support (FAPERJ) of Brazil. E.Y.J. acknowledges support by
Cancer Research UK (A10976) and by the Wellcome Trust Centre for Human
Genetics (090532/Z/09/Z). X.H. acknowledges support by NIH (RO1-
GM057603) and by Boston Children’s Hospital Intellectual and Developmental
Disabilities Research Center (P30 HD-18655). X.H. is an American Cancer So-
ciety Research Professor.
Received: January 8, 2015
Revised: February 17, 2015
Accepted: February 17, 2015
Published: March 12, 2015
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